Project Summary - Type 2 diabetes (T2D) plagues nearly 10% of the US population (~30 million people); a shocking 84 million more have prediabetes and show signs of impaired glucose tolerance. Compounding this problem, certain insulin-sensitizing drugs are undergoing global market suspensions, leaving prediabetic and T2D patients with few treatment options and creating an urgent need for new therapeutics. Because multi- tissue dysfunction contributes to prediabetes and progression to T2D, prevention or reversal of these diseases requires a multi-pronged approach. Specifically, pancreatic ?-cell dysfunction and skeletal muscle (skm) insulin resistance are primary features of human prediabetes and T2D. STX4, a primary component of the SNARE exocytosis machinery, is reduced in ?-cells and skm from humans with T2D. Furthermore, STX4 is required for normal ?-cell insulin secretion and skm insulin sensitivity in mouse models and human islets. Our long-term goal is to understand how ?-cell and skm signaling can be manipulated to prevent or reverse prediabetes and halt the progression to T2D. Our central hypothesis is that STX4 enrichment supports ?-cells and/or skeletal muscle to prevent and reverse the damaging effects of diabetogenic stress. The rationale for the proposed research is that once these new mechanisms of STX4 are elucidated, STX4 signaling can be manipulated to prevent or reverse T2D. During the last funding cycle, we revealed new roles for STX4 in promoting ?-cell survival and skm function. Indeed, mice overexpressing STX4 in these tissues have a remarkable 33% extended lifespan and are protected from aging- and diet-induced metabolic dysfunction. Also, we showed that STX4 can reverse insulin resistance in obese mice. However, the tissue-specific role of STX4 was unknown. Therefore, we generated inducible ?-cell- and skm- specific STX4 overexpression and knockout mice, as well as RNA-based candidate therapeutics for tissue-specific STX4 enrichment. Our provocative new preliminary data indicate that STX4 participates in anti-inflammatory signaling in the ?-cells, and localizes to the outer mitochondrial membrane to regulate mitochondrial function in skm. Therefore, the objective of this application is to test these candidate mechanisms linking STX4 enrichment and protection of ?-cells and skm from diabetogenic stress and to evaluate candidate STX4 enrichment therapeutics. We will use our inducible mouse models and human tissues/cells for these studies. In Aim 1, we will evaluate the mechanisms underlying the protective actions of STX4 in ?-cells and test novel ?-cell STX4 enrichment strategies; in Aim 2, we will delineate how STX4 enrichment protects skm and how it reverses HFD-induced dysfunction. We will use innovative molecular tools to test novel hypotheses about STX4 action in the context of a translation-focused institutional environment at City of Hope. This work will positively impact diabetes research by evaluating a promising candidate strategy to reverse prediabetes and halt progression to T2D and by uncovering novel mechanisms of glycemic regulation.